| 5-氮胞苷对大鼠脂肪间充质干细胞的诱导分化作用及相关蛋白的表达 |
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| 引用本文： | 夏菁,徐丽艳,刘雪兰,方建江,徐少博,陈静静.5-氮胞苷对大鼠脂肪间充质干细胞的诱导分化作用及相关蛋白的表达[J].中华全科医学,2019,17(4):557-560. |
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| 作者姓名： | 夏菁 徐丽艳 刘雪兰 方建江 徐少博 陈静静 |
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| 作者单位： | 宁波市医疗中心李惠利医院急诊科, 浙江 宁波 315014 |
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| 基金项目： | 浙江省医药卫生科技计划项目(2014KYB229) |
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| 摘 要： | 目的 探讨5-氮胞苷(5-Aza)对大鼠脂肪间充质干细胞(ADMSCs)的诱导分化作用及相关蛋白的表达。 方法 采取酶消化法和贴壁法分离培养Sprague-Dawley(SD)大鼠腹部皮下脂肪组织，采用抗单核细胞人类白细胞抗原DR(HLA-DR)、兔抗CD13、CD44、CD105、血管性血友病因子(vWF)多克隆抗体对培养细胞进行鉴定，排除造血干细胞及成纤维细胞，取第3代ADMSCs细胞，以每孔1×104个细胞接种于24孔培养板中，并将其随机分为正常组和诱导组，正常组细胞于常规培养基中加入无菌生理盐水培养，诱导组细胞于常规培养基中加入浓度为10 μmol/L的5-Aza进行诱导培养。比较2组细胞的形态结构、心肌肌钙蛋白T(cTnT)、心肌肌钙蛋白I(cTnI)及缝隙连接蛋白43(Connexin43)基因表达量及cTnT、cTnI、Connexin43蛋白的表达情况。 结果 诱导组ADMSCs细胞增殖缓慢，体积增大并逐渐变为棒状或宽梭形，形态呈心肌细胞特征；RT-qPCR结果显示，诱导组细胞cTnT、cTnI及Connexin43基因表达量增强；免疫组化结果显示，诱导组细胞细胞质明显可见棕黄色或棕褐色颗粒物质，而正常组细胞细胞质中棕黄色或棕褐色颗粒物质较少，且诱导组细胞cTnT、cTnI及Connexin43蛋白阳性表达评分明显高于正常组(P<0.05)。 结论 大鼠脂肪组织中含有多向分化潜能的间充质干细胞(MSCs)，5-Aza可以诱导大鼠ADMSCs向心肌细胞分化，ADMSCs有望成为治疗心肌梗死的种子细胞。
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| 关 键 词： | 5-氮胞苷 大鼠脂肪间充质干细胞 诱导 分化 心肌样细胞 |
| 收稿时间： | 2018-10-17 |
Differentiation-inducing effect of 5-azacytidine in rat adipose-derived mesenchymal stem cells and the expressions of related protein |
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| Affiliation: | Emergency Department, Ningbo Medical Center Lihuili Hospital, Ningbo, Zhejiang 315014, China |
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| Abstract: | Objective To investigate the differentiation inducing effect of 5-Azacytidine (5-Aza) in rat adipose-derived mesenchymal stem cells (ADMSCs) and the expressions of related protein. Methods The abdominal subcutaneous adipose tissue of Sprague-Dawley (SD) rat was isolated and cultured by enzyme digestion and adhesion method. The cultured cells were identified by using anti-monocyte human leukocyte antigen DR (HLA-DR), rabbit anti-CD13, CD44, CD105 and von willebrand factor (vWF) polyclonal antibody. The hematopoietic stem cells and fibroblasts were excluded. The third passage ADMSCs cells were used to inoculate into 24 well culture plates with the concentration of 1×104 cells/well. And the cultured cells were randomly divided into normal group and induction group, the cells in normal group was cultured in normal saline with sterile saline, the cells in induction group was incubated with 5-Aza at a concentration of 10 μmol/L. The morphological changes, cardiac troponin T (cTnT), cardiac troponin I (cTnI) and Connexin43 mRNA expression and cTnT, cTnI, Connexin43 protein expression were compared between the two groups. Results The proliferation of ADMSCs cells in the induction group was slowly, their volume was increased and gradually changed into a rod-shaped or wide-spindle shape, the morphology was characterized by cardiomyocytes; RT-qPCR results showed that the mRNA expression of cTnT, cTnI and Connexin43 were enhanced in the induced group; Immunohistochemistry results showed that the brownish yellow or chocolate brown granular matters were obviously visible in the cytoplasm of induction group, there were fewer brownish yellow or chocolate brown granular matters in the cytoplasm of the normal group, and the protein positive expression scores of cTnT, cTnI and Connexin43 in the induced group was significantly higher than that of the normal group (P<0.05). Conclusion Rat adipose tissue contains mesenchymal stem cells (MSCs) with multi-directional differentiation potential, 5-Aza can induce rat ADMSCs to differentiate into cardiomyocytes cells, and ADMSCs are expected to become the seed cells for the treatment of myocardial infarction. |
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